Cisplatin is a potent chemotherapeutic agent widely used to treat malignant neoplasms. Side effects, like ototoxicity compromise the quality of life of cancer survivors. Cisplatin increases formation of reactive oxygen species (ROS) in the cochlea. A unique isoform of NADPH oxidase, NOX-3, is present in rat cochlea. This enzyme generates superoxide radicals that can damage outer hair cells (OHCs), leading to hearing loss. ROS can activate cochlear genes that may be protective or harmful. Protective molecules may include a novel protein, kidney injury molecule (KIM)-1. It is induced in kidney by cisplatin. This molecule may enhance repair and regeneration of proximal tubule cells. Our laboratory was the first to show KIM-1 in the cochlea and its induction by cisplatin. ROS may up-regulate KIM-1. It is unknown whether this protein protects the cochlea from cisplatin. ROS resulting from cisplatin may also increase expression of potentially harmful transient receptor potential vanilloid 1 receptors (TRPV1). Activation of NOX-3 and TRPV1 may overwhelm cochlear antioxidant defenses, leading to cell death. Experiments in this application seek to more precisely define mechanisms of cisplatin ototoxicity in order to find ways to minimize toxicity. Studies proposed will utilize a cochlear cell line (UB/OC-1) and parallel studies in rats to address three specific aims to: 1) investigate mechanisms of NOX-3 activation by cisplatin in the cochlea; 2) determine whether expression of KIM-1 confers protection against cisplatin ototoxicity and to determine potential mechanisms of its induction; and 3) study whether TRPV1 contributes to cisplatin ototoxicity and to determine mechanisms of its induction. For aim 1, we will administer systemically the antioxidant lipoic acid or short-interfering RNAs (siRNAs) on the round window (RW) to block activation of NOX-3 by cisplatin. In aim 2, we will use RW application of inhibitors of signaling molecules to see if they are involved in KIM-1 induction; and RW application of siRNA for KIM-1 to see if cisplatin ototoxicity increases. For aim 3, we will investigate whether RW application of the TRPV1 antagonist, capsazepine, protects against cisplatin ototoxicity. We will also examine effects of RW administration of siRNA against TRPV1 to see whether this prevents cisplatin ototoxicity. This research could provide new insights into mechanisms of cisplatin ototoxicity and novel therapeutic approaches to ameliorate cisplatin ototoxicity. Cisplatin causes hearing loss in a large percentage of cancer patients who receive this drug to cure their cancer. It would be of great benefit to cancer survivors if a treatment could be discovered that would prevent hearing loss without interfering with the cancer killing effects of cisplatin. This research seeks to discover new potential treatments to reduce the side effect of hearing loss resulting from the treatment of cancer with the drug cisplatin. Cisplatin causes hearing loss in a large percentage of cancer patients who receive this drug to cure their cancer.